Somatic cells have a finite proliferative capacity. The role of telomeres in regulating cell division, in particular the number of cell divisions a cell lineage can undergo, is critical. Telomeres are repetitive DNA sequences, typically G-rich on one strand (C-rich on the complementary strand), at the termini of linear chromosomes. In human (and indeed most vertebrate) chromosomes telomeres comprise typically several thousand copies of the sequence (5′-TTAGGG-3′)n. Typically, telomeres shorten with each round of cell division, at least in part due to the incomplete replication of the ends of linear chromosomes. When telomeres become too short this evokes normal cellular DNA damage repair pathways. A complex series of biochemical and morphological changes ensues resulting in cell cycle arrest and cell death, either via replicative senescence or via programmed cell death such as apoptosis. Senescence and apoptosis each constitute major pathways for the regulation of cell proliferation. These processes are beneficial, for example, in the suppression of tumorigenesis and limiting disease progression more generally (see, for example, Collado et al., 2005). In pathological conditions characterised by aberrant cellular proliferation, such as cancer, the normal senescence and apoptotic pathways are circumvented enabling cells to become immortal.
Broadly speaking, two alternative telomere maintenance mechanisms are used by cancer cells to counteract the innate telomere loss that normally accompanies linear chromosome replication. The first involves synthesis of new telomeric DNA from an RNA template using the reverse transcriptase telomerase. Alternatively, telomeres may be maintained via a telomerase-independent process known as Alternative Lengthening of Telomeres (ALT) (Bryan et al., 1995; Bryan et al., 1997). The ALT mechanism involves recombination-dependent DNA replication using either the same telomere or another telomere, or possibly extrachromosomal telomeric DNA as the copy template (see, for example, Henson et al., 2002). ALT generates sudden, large increases in telomere length, consistent with either a long linear telomeric template or a rolling mechanism, such as rolling circle amplification (RCA).
Cells of a number of human tumours utilize ALT, especially those arising in brain, bone and connective tissue (see, for example, Bryan et al., 1997; Hakin-Smith et al., 2003; Henson et al., 2005; Ulaner et al., 2003; Villa et al., 2008), but the full extent of the role and importance of ALT in many cancers is yet to be fully elucidated. The prognosis for patients with an ALT[+] cancer is generally poor, with median survival ranging from 2 to 5 years.
Both telomerase and ALT represent attractive targets for anti-cancer treatment and there is an increasing recognition that for many cancers it will be desirable to have at our disposal therapies that are specific for ALT[+] cells and therapies that are specific for telomerase[+] cells. Cancer cells of a specific cancer type in one individual may utilise telomerase for telomere maintenance, whereas cells of the same cancer type in another individual may utilise ALT, and indeed cells within a single tumour may utilise different telomere maintenance mechanisms. It is also thought that cancer cells may have the capacity under certain conditions to switch between telomerase-induced telomere maintenance and ALT, raising the possibility that as telomerase-specific cancer therapies increase in clinical use, the prevalence of ALT[+] cancer cells may increase. Thus, with the development of ALT-specific and telomerase-specific therapies, it will become increasingly important to determine whether the cancer in any given individual is ALT[+] or telomerase[+].
There also remains the need for the identification and development of suitable therapeutic agents capable of inhibiting ALT. There is also a need for the identification and development of activators of ALT to induce cellular immortality and for application in, for example, research into aging. However hampering such efforts is that there is presently no enzyme activity or protein that is known to be specific for ALT.
To date, ALT activity has been demonstrated indirectly, for example by the maintenance of average telomere length over many population doublings in the absence of telomerase. The presence of ALT has also been deduced by observing rapid changes in the length of individual telomeres or other characteristics of ALT[+] cells, the presence of a highly heterogeneous telomere length distribution, increased telomeric recombination, and telomeric DNA in promyelocytic leukemia nuclear bodies. None of the current assays for ALT activity are suitable as a screen for ALT inhibitors, which requires a simple, definitive assay that is rapidly and linearly responsive to changes in ALT activity. Detection of ALT activity in cancer patients currently requires the availability of tumour specimens or biopsies, and the techniques available lack responsiveness and/or sensitivity, and are not generally suitable for use in routine pathology laboratories.
There is a clear need for the development of accurate, reliable and rapidly responsive assays of ALT activity, based on parameters that are specific for ALT[+] cells. As disclosed herein the present inventors have found that the presence of partially double-stranded telomeric DNA circles is a highly specific and quantitative marker for an active ALT mechanism within a cell.